Neurospora : lure and lore.

The mutant has a unique phenotype. It arrests sexual development when the fruiting bodies (perithecia) attain only 40% of their normal diameter, regardless of whether the mutant participates in a cross with the wild type ( x ) as the male or female parent. I first learnt about when this journal invited me to review '' by D. D. Perkins, A. Radford and M. S. Sachs ( 80: 53-54, 2001). The compendium also informed me that the first Neurospora genetic map was published here ( 32, 243-256, 1936). The mutant was discovered and characterized by T. E. Johnson, who also localized the mutation to a chromosome 1 segment that spanned more than 3.3 Mb DNA ( 92, 1107-1120, 1979). The second paper came 30 years later from my laboratory. We mapped the mutation to a single base pair, a T:A to A:T transversion mutation, and thus identified the altered gene ( 88: 33-39, 2009). To map , we leveraged our expertise in making strains bearing chromosome segment duplications. The strains were generated in crosses of the wild type with translocation strains ( x ). A translocation transfers a segment of one chromosome into another. Mapping with s localized to a 330 kbp segment. Conventional mapping with crossovers and selection against noncrossovers subsequently localized it to a 33 kbp segment. This interval was small enough to pick up the mutation by sequencing its DNA. The Fmf-1 protein activates genes required for mating pheromone signalling. The male gametes (conidia) fail to secrete the pheromone that attracts receptors on the female sexual structures (protoperithecia). Conversely, protoperithecia do not express the cognate receptor for the pheromone from the conidia. Consequently, the x cross fails to fertilize protoperithecia and arrests their maturation into perithecia. Genetic mapping, especially mapping, fails to impress many nongeneticists these days. How do x crosses produce progeny? Why are s and crossovers even needed? Why select against noncrossovers? Why not just sequence the genomes of the wild type and mutant, identify genes whose DNA is altered in the mutant, and then test them one by one? Many forget that DNA sequencing, especially of 'hard to access' centromeric sequences, was not as easy and inexpensive then. Isolating offered us the possibility of enriching for RIP-defective mutants. RIP is a mutational process that occurs during a sexual cross and induces multiple G:C to A:T transition mutations in all copies of any DNA sequences duplicated in the otherwise haploid Neurospora genome. It is the most mutagenic process known in biology. Reputedly, linked duplications were 'mutated at frequencies of 95% or more' ( 75: 313-324, 1996). My student, Srividhya Iyer, created a linked duplication of by inserting a second copy of it within 5 kbp of the endogenous gene. Most progeny from duplication-homozygous crosses would inherit a RIP-mutated allele, rendering them infertile. If the f progeny are germinated en masse, and allowed to randomly inter-cross, then only crosses between the minor fraction of non-RIPed progeny can generate the f. Likewise, for the f, f, etc. Later generations, hence, become progressively enriched for RIP-defective mutants. In the f progeny examined by Iyer, the RIP-induced mutant fraction was not 95%, but 'merely' 85%, a lesser enrichment efficiency than we desired. Therefore, the enrichment attempt was abandoned. This is not for the first time, nor the last, that a beautiful strategy was killed by an ugly fact.